The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

Abstract

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.Lactococcus lactis is a lactic acid bacterium that is used as a starter culture in the dairy industry. L. lactis has a rather simple and well-characterized metabolism and converts sugars mainly into lactic acid. In recent years L. lactis has also been evaluated as organism for production of industrial lactic acid (23, 38) and has been subjected to metabolic engineering to reroute its metabolism towards novel products (13,18,22,25,26). To take full advantage of bacterial processes, it is very important to understand which intracellular and extracellular factors influence the metabolic rate and product formation. L. lactis is an attractive model organism for studies of glycolysis and pyruvate metabolism, since oxidative phosphorylation normally does not occur and more than 90% of the carbon source is recovered as fermentation by-products, mainly lactate. In glycolysis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) converts NAD ϩ to NADH, which must be regenerated for continued carbon catabolism. Lactate dehydrogenase (LDH) regenerates NAD ϩ by converting the end product of glycolysis, pyruvate, to lactate. An alternative way for lactococci to regenerate NAD ϩ is by production of ethanol by alcohol dehydrogenase (ADH...

Kinetic Analysis of Bifidobacterial Metabolism Reveals a Minor Role for Succinic Acid in the Regeneration of NAD + through Its Growth-Associated Production

“…Several factors related to the specific sugar consumption rate, such as the sugar uptake systems and the conversions necessary to incorporate a specific sugar into the fructose-6-phosphate pathway, can have great effects on which metabolic route is used (3,17). It has also been reported that in L. lactis the NADH ϩ H ϩ /NAD ϩ ratio (11) or the pool of ADP and ATP (30) can have an effect on changes in end product formation. However, the fact that B. longum BB536 had almost the same specific rate of sugar consumption on fructose and on lactose, although the amounts and ratios of the end products differed, proved that the specific sugar consumption rate was not the only determining factor.…”

Phosphoketolase Pathway Dominates in Lactobacillus reuteri ATCC 55730 Containing Dual Pathways for Glycolysis

“…Triethanolamine buffer (50 mM, pH 7.0) with MgCl 2 (10 mM) was used in all assays. The method used for the inhibition study of phosphoglucose isomerase (PGI) by 6-phosphogluconate was similar to the method of Richter et al (22) except that the perchloric acid treatment step was done according to the method of Palmfeldt et al (19). (3) was used in the analysis for genes known to be involved in sucrose and glucose metabolism.…”

“…Sucrose, glucose, fructose, and mannitol were separated on an Aminex HPX-87C column (Bio-Rad, Hercules, CA) at 85°C with Milli-Q water as the mobile phase (19).…”

Effect of Perturbation of ATP Level on the Activity and Regulation of Nitrogenase in Rhodospirillum rubrum

“…Nevertheless, there has been no direct in vivo test of the impact of changes in internal ATP levels on P II function, and the focus of the present study was to measure that impact by monitoring the DRAT/DRAG system. ATP plays many critical roles in living cells (3,7,11,13), and there are clearly cases where absolute ATP level is also an important indicator of the physiological state in prokaryotes (54,55,59,60). Schneider et al reported that the changes in ATP concentration affect the transcription of the rrnB P1 promoter (59).…”